Transcription factors can modulate gene expression, either increasing or decreasing (inducing or repressing) the rate of transcription. This modulation results in differential levels of gene expression at various developmental stages, in different growth phases and cell types, and in response to different exogenous (e.g., environmental) and endogenous stimuli throughout the life cycle of the organism. Because transcription factors are key controlling elements of biological pathways, altering the expression levels of one or more transcription factors can change entire biological pathways in an organism.
Transcriptional regulation of most eukaryotic genes occurs through the binding of transcription factors to sequence specific binding sites in their promoter regions. Many of these protein binding sites have been conserved through evolution and are found in the promoters of diverse eukaryotic organisms. One such feature that shows a high degree of conservation is the CCAAT-box (Edwards et al, Plant Physiol. 117:1015-1022, 1998). The CCAAT family of transcription factors, also be referred to as the “CART”, “CAAT-box” or “CCAAT-box” family, are characterized by their ability to bind to a CCAAT-box element in the upstream region of a gene, typically located 80 to 300 bp 5′ from a transcription start site (Gelinas et al., Nature 313:323-325, 1985). This cis-acting regulatory element is found in all eukaryotic species and is estimated to be present in the promoter and/or enhancer regions of approximately 30% of genes (see, e.g. Bucher and Trifonov, J. Biomol. Struct. Dyn. 5: 1231-1236, 1988; Bucher, J. Mol. Biol. 212:563-578, 1990). The CCAAT-box element can function in either orientation, and can operate alone or in cooperation with other cis regulatory elements (Tasanen et al., J. Biol. Chem. 267:11513-11519, 1992).
CCAAT-box binding proteins constitute a large family of transcription factors first identified in yeast and named HAP for Heme-Activation Protein. They combine to form a heteromeric protein complex that activates transcription by binding to CCAAT boxes in eukaryotic promoters. In plants, CCAAT binding transcription factors are thought to bind DNA as heterotrimers composed of HAP2-like, HAP3-like and HAP5-like subunits. The HAP heterotrimer is also referenced in the scientific literature as the CCAAT box binding factor (CBF) or Nuclear Factor Y (NF-Y), which comprises an NF-YA subunit (corresponding to the HAP2-like subunit), an NF-YB subunit (corresponding to the HAP3-like subunit) and an NF-YC subunit (corresponding to the HAP5-like subunit) (Mantovani et al., Nucl. Acids Res. 20: 1087-1091, 1992; Mantovani, Gene 239:15-27, 1999; Gusmaroli et al., Gene 264:173-185, 2001; Gusmaroli et al., Gene 283:41-48, 2002). HAP2-, HAP3- and HAP5-like proteins have two highly conserved sub domains, one that functions in subunit interaction and the other that acts in a direct association with DNA. Outside of these two regions, HAP-like proteins can be quite divergent in sequence and in overall length. Throughout the disclosure, the HAP terminology is used for the NF-YB subunit, and in particular, the term “HAP3-like protein” or “HAP3 protein” is used, but other names such as CBF-A and NF-YB are interchangeable and denote the same protein. The NF-Y terminology is most commonly used herein for HAP3 partners, for example, and its transcription factor complex partners of HAP3 (NF-YB) are referred to herein as “NF-YA” (HAP2) and “NF-YC (HAP5)”.
In yeast, there is a single gene for each HAP subunit (e.g., HAP2, HAP3, and HAP5), and the HAP proteins are involved in the transcriptional control of metabolic processes such as the regulation of catabolic derepression of cycl and other genes involved in respiration (Becker et al., Proc. Natl. Acad. Sci. USA 88:1968-1972, 1991). In contrast, multiple forms of each HAP homolog have been identified in plants (Edwards et al, 1998, supra; Gusmaroli et al., 2002, supra). The general domain structure of HAP3-like proteins has been documented in great detail (see, e.g. U.S. Pat. No. 7,868,229; Lotan et al., Cell 93:1195-1205, 1998). HAP3-like proteins contain an amino-terminal A domain, a central B domain and a carboxy-terminal C domain. There is very little sequence similarity between different HAP3-like protein family members (paralogs) in the A and C domains; it is therefore widely assumed that the A and C domains could provide a degree of functional specificity to each member of the HAP3-like protein subfamily.
Generally, HAP3-like proteins comprise a “conserved protein-protein and DNA-binding interaction module” within their histone fold motif or “HFM” (Gusmaroli et al., Gene 283:41-48, 2002). The HFM, which is reported to be required for HAP function (Edwards et al., Plant Physiol. 117:1015-1022, 1998), is within the larger highly conserved B domain (Lee et al., Proc. Natl. Acad. Sci. 100: 2152-2156, 2003) which is responsible for both DNA binding and subunit association. According to Gusmaroli et al., 2002, supra “all residues that constitute the backbone structure of the HFMs are conserved, and residues such as AtNF-YB-10 [At3g53340; an Arabidopsis HAP3-like protein] N38, K58, and Q62, involved in CCAAT-binding, and E67 and E75, involved in NF-YA association (Maity and de Crombrugghe, Trends Biochem Sci. 23:174-178, 1998; Zemzoumi et al., J. Mol. Biol. 286:327-337, 1999), are maintained”.
Leafy cotyledon1 (LEC1), one of ten HAP3-like proteins encoded by the Arabidopsis thaliana genome, has been identified as a central regulator that affects embryogenesis (as does the related “LEC1-like” or “L1L” protein (Kwong et al. The Plant Cell 15:5-18, 2003) and oil accumulation in maize embryos (U.S. Pat. No. 7,294,759). Like other HAP3-like proteins, LEC1 has three domains: an amino terminal A domain, a central B domain, and a carboxyl terminal C (Harada et al., Proc. Natl. Acad. Sci 100(4): 2152-2156, 2003). The B domain typically includes about 90 residues and often has a conserved signature sequence of 7 residues of Met Pro Ile Ala Asn Val Ile (MPIANVI), sometimes referred to as the PIANO motif. The LEC1 and L1L proteins also have sixteen conserved amino acids within the B domain that differ from the amino acids at the same positions of the B domain in other HAP3-like proteins, which are known as the “non-LEC1-type” HAP3-like proteins (Kwong et al., 2003, supra; Lee et al., 2003, supra). Molecular and genetic analysis revealed non-LEC1 like HAP3-like protein family members of higher plants to be involved in the control of diverse biological processes including drought tolerance (Nelson et al. Proc. Natl. Acad. Sci 104: 16450-16455) and timing of flowering (U.S. Pat. No. 7,868,229).
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